Parallel Epigenomic and Transcriptomic Responses to Viral Infection in Honey Bees ()

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Honey bees are a critical pollinator of a wide variety of agricultural crops, but beekeepers experience heavy annual losses of honey bee colonies. Several factors are associated with colony losses, in particular infection with Israeli Acute Paralysis Virus (IAPV). Despite the importance of viruses to honey bee health, our understanding of the molecular mechanisms mediating host-pathogen interactions is limited. Here, we characterized the epigenomic and transcriptomic responses of honey bees to short term (<24 hour) IAPV infection. We found significant expression differences in 753 genes between infected and control bees, including genes involved in immune and epigenetic pathways. However, IAPV-regulated genes did not overlap significantly with genes that respond to bacterial or microsporidian infection, suggesting that honey bees utilize distinct pathways for different immune challenges. Additionally, DNA methylation status of 156 genes changed significantly, including several genes that have been linked to antiviral immune responses in humans. Interestingly, there was no significant overlap between the differentially methylated and differentially expressed genes, suggesting that honey bees may possess a parallel genomic mechanisms to respond to viral infection.

Vyšlo v časopise: Parallel Epigenomic and Transcriptomic Responses to Viral Infection in Honey Bees (). PLoS Pathog 11(3): e32767. doi:10.1371/journal.ppat.1004713
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004713

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1. Potts SG, Biesmeijer JC, Kremen C, Neumann P, Schweiger O, Kunin WE. Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol. Elsevier Ltd; 2010;25: 345–53. doi: 10.1016/j.tree.2010.01.007 20188434

2. Steinhauer N a, Rennich K, Wilson ME, Caron DM, Lengerich EJ, Pettis JS, et al. A national survey of managed honey bee 2012–2013 annual colony losses in the USA: results from the Bee Informed Partnership. J Apic Res. 2014;53: 1–18. doi: 10.3896/IBRA.1.53.1.01

3. Klein A-M, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham S a, Kremen C, et al. Importance of pollinators in changing landscapes for world crops. Proc Biol Sci. 2007;274: 303–13. doi: 10.1098/rspb.2006.3721 17164193

4. Yang X, Cox-Foster DL. Impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification. Proc Natl Acad Sci U S A. 2005;102: 7470–5. doi: 10.1073/pnas.0501860102 15897457

5. DeGrandi-Hoffman G, Chen Y, Huang E, Huang MH. The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (Apis mellifera L.). J Insect Physiol. Elsevier Ltd; 2010;56: 1184–91. doi: 10.1016/j.jinsphys.2010.03.017 20346950

6. Di Prisco G, Cavaliere V, Annoscia D, Varricchio P, Caprio E, Nazzi F, et al. Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees. Proc Natl Acad Sci U S A. 2013;110: 18466–71. doi: 10.1073/pnas.1314923110 24145453

7. Chen YP, Siede R. Honey bee viruses. Adv Virus Res. 2007;70: 33–80. doi: 10.1016/S0065-3527(07)70002-7 17765703

8. Runckel C, Flenniken ML, Engel JC, Ruby JG, Ganem D, Andino R, et al. Temporal analysis of the honey bee microbiome reveals four novel viruses and seasonal prevalence of known viruses, Nosema, and Crithidia. PLoS One. 2011;6: e20656. doi: 10.1371/journal.pone.0020656 21687739

9. Bailey L, Ball B V. Honey Bee Pathology. 2nd ed. London: Academic Press INC.; 1991.

10. Iqbal J, Mueller U. Virus infection causes specific learning deficits in honeybee foragers. Proc Biol Sci. 2007;274: 1517–21. doi: 10.1098/rspb.2007.0022 17439851

11. Maori E, Paldi N, Shafir S, Kalev H, Tsur E, Glick E, et al. IAPV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion. Insect Mol Biol. 2009;18: 55–60. doi: 10.1111/j.1365-2583.2009.00847.x 19196347

12. Dainat B, Evans JD, Chen YP, Gauthier L, Neumann P. Predictive markers of honey bee colony collapse. PLoS One. 2012;7: e32151. doi: 10.1371/journal.pone.0032151 22384162

13. Genersch E, von Der Ohe W, Kaatz H, Schroeder A, Otten C, Buchler R, et al. The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie. 2010;41: 332–352.

14. Van Dooremalen C, Gerritsen L, Cornelissen B, van der Steen JJM, van Langevelde F, Blacquière T. Winter survival of individual honey bees and honey bee colonies depends on level of Varroa destructor infestation. PLoS One. 2012;7: e36285. doi: 10.1371/journal.pone.0036285 22558421

15. De Miranda JR, Cordoni G, Budge G. The Acute bee paralysis virus-Kashmir bee virus-Israeli acute paralysis virus complex. J Invertebr Pathol. Elsevier Inc.; 2010;103 Suppl: S30–47. doi: 10.1016/j.jip.2009.06.014 19909972

16. Boncristiani HF, Evans JD, Chen Y, Pettis J, Murphy C, Lopez DL, et al. In vitro infection of pupae with Israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (Apis mellifera). PLoS One. 2013;8: e73429. doi: 10.1371/journal.pone.0073429 24039938

17. Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran N a, et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 2007;318: 283–7. doi: 10.1126/science.1146498 17823314

18. Hou C, Rivkin H, Slabezki Y, Chejanovsky N. Dynamics of the presence of israeli acute paralysis virus in honey bee colonies with colony collapse disorder. Viruses. 2014;6: 2012–27. doi: 10.3390/v6052012 24800677

19. Vanengelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, Nguyen BK, et al. Colony collapse disorder: a descriptive study. PLoS One. 2009;4: e6481. doi: 10.1371/journal.pone.0006481 19649264

20. Cornman RS, Tarpy DR, Chen Y, Jeffreys L, Lopez D, Pettis JS, et al. Pathogen webs in collapsing honey bee colonies. PLoS One. 2012;7: e43562. doi: 10.1371/journal.pone.0043562 22927991

21. Chen YP, Pettis JS, Corona M, Chen WP, Li CJ, Spivak M, et al. Israeli Acute Paralysis Virus: Epidemiology, Pathogenesis and Implications for Honey Bee Health. PLoS Pathog. 2014;10: e1004261. doi: 10.1371/journal.ppat.1004261 25079600

22. Merkling SH, van Rij RP. Beyond RNAi: antiviral defense strategies in Drosophila and mosquito. J Insect Physiol. 2013;59: 159–70. doi: 10.1016/j.jinsphys.2012.07.004 22824741

23. Wang X, Aliyari R, Li W-X, Li H-W, Kim K, Carthew R, et al. RNA interference directs innate immunity against viruses in adult Drosophila. Science. 2006;312: 452–4. doi: 10.1126/science.1125694 16556799

24. Flenniken ML, Andino R. Non-specific dsRNA-mediated antiviral response in the honey bee. PLoS One. 2013;8: e77263. doi: 10.1371/journal.pone.0077263 24130869

25. Liu X, Zhang Y, Yan X, Han R. Prevention of Chinese sacbrood virus infection in Apis cerana using RNA interference. Curr Microbiol. 2010;61: 422–8. doi: 10.1007/s00284-010-9633-2 20379718

26. Desai SD, Eu Y-J, Whyard S, Currie RW. Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion. Insect Mol Biol. 2012;21: 446–55. doi: 10.1111/j.1365-2583.2012.01150.x 22690671

27. Ryabov E V, Wood GR, Fannon JM, Moore JD, Bull JC, Chandler D, et al. A virulent strain of deformed wing virus (DWV) of honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission. PLoS Pathog. 2014;10: e1004230. doi: 10.1371/journal.ppat.1004230 24968198

28. Finnegan EJ, Genger RK, Peacock WJ, Dennis ES. Dna methylation in plants. Annu Rev Plant Physiol Plant Mol Biol. 1998; 223–247.

29. Goodwin J, Chapman K, Swaney S, Parks TD, Wernsman E, Dougherty W. Genetic and Biochemical Dissection of Transgenic RNA-Mediated Virus Resistance. Plant Cell. 1996;8: 95–105. 8597662

30. Robertson KD. DNA methylation and human disease. Nat Rev Genet. 2005;6: 597–610. doi: 10.1038/nrg1655 16136652

31. Fang J, Hao Q, Liu L, Li Y, Wu J, Huo X, et al. Epigenetic changes mediated by microRNA miR29 activate cyclooxygenase 2 and lambda-1 interferon production during viral infection. J Virol. 2012;86: 1010–20. doi: 10.1128/JVI.06169-11 22072783

32. Kathiria P, Sidler C, Golubov A, Kalischuk M, Kawchuk LM, Kovalchuk I. Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants. Plant Physiol. 2010;153: 1859–70. doi: 10.1104/pp.110.157263 20498336

33. Li W, Sun W, Liu L, Yang F, Li Y, Chen Y, et al. IL-32: a host proinflammatory factor against influenza viral replication is upregulated by aberrant epigenetic modifications during influenza A virus infection. J Immunol. 2010;185: 5056–65. doi: 10.4049/jimmunol.0902667 20889550

34. Mukherjee S, Vipat VC, Chakrabarti AK. Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes. Influenza Other Respi Viruses. 2013;1: 1–8. doi: 10.1111/irv.12127

35. Takacs M, Segesdi J, Banati F, Koroknai A, Wolf H, Niller HH, et al. The importance of epigenetic alterations in the development of epstein-barr virus-related lymphomas. Mediterr J Hematol Infect Dis. 2009;1: e2009012. doi: 10.4084/MJHID.2009.012 21416002

36. Tang B, Zhao R, Sun Y, Zhu Y, Zhong J, Zhao G, et al. Interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsRNA treatment. Mol Immunol. Elsevier Ltd; 2011;48: 1001–8. doi: 10.1016/j.molimm.2011.01.003 21353307

37. Urieli-Shoval S, Gruenbaum Y, Sedat J, Razin A. The absence of detectable methylated bases in Drosophila melanogaster DNA. 1982;146: 148–152. 6814955

38. Capuano F, Kok R, Blom HJ, Ralser M. Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species. Anal Chem. 2014; 3697–3702. doi: 10.1021/ac500447w 24640988

39. Wang Y, Jorda M, Jones PL, Maleszka R, Ling X, Hugh M, et al. Functional System CpG Methylation in a Social Insect. 2006;314: 645–647. 17068262

40. Suzuki MM, Bird A. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet. 2008;9: 465–76. doi: 10.1038/nrg2341 18463664

41. Zemach A, McDaniel IE, Silva P, Zilberman D. Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science. 2010;328: 916–9. doi: 10.1126/science.1186366 20395474

42. Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31: 89–97. doi: 10.1016/j.tibs.2005.12.008 16403636

43. Foret S, Kucharski R, Pellegrini M, Feng S, Jacobsen SE, Robinson GE, et al. DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees. Proc Natl Acad Sci U S A. 2012;109: 4968–73. doi: 10.1073/pnas.1202392109 22416128

44. Kucharski R, Maleszka J, Foret S, Maleszka R. Nutritional control of reproductive status in honeybees via DNA methylation. Science. 2008;319: 1827–30. doi: 10.1126/science.1153069 18339900

45. Bird AP. Gene number, noise reduction and biological complexity. Trends Genet. 1995;11: 94–100. doi: 10.1016/S0168-9525(00)89009-5 7732579

46. Huh I, Zeng J, Park T, Yi S V. DNA methylation and transcriptional noise. Epigenetics Chromatin. 2013;6: 9. doi: 10.1186/1756-8935-6-9 23618007

47. Fussnecker BL, McKenzie AM, Grozinger CM. cGMP modulates responses to queen mandibular pheromone in worker honey bees. J Comp Physiol A Sensory, Neural, Behav Physiol. 2013;197: 939–948. doi: 10.1007/s00359-011-0654-5 cGMP

48. Li Z, Chen Y, Zhang S, Chen S, Li W, Yan L, et al. Viral infection affects sucrose responsiveness and homing ability of forager honey bees, Apis mellifera L. PLoS One. 2013;8: e77354. doi: 10.1371/journal.pone.0077354 24130876

49. Hunter W, Ellis J, Vanengelsdorp D, Hayes J, Westervelt D, Glick E, et al. Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae). PLoS Pathog. 2010;6: e1001160. doi: 10.1371/journal.ppat.1001160 21203478

50. Richard F-J, Tarpy DR, Grozinger CM. Effects of insemination quantity on honey bee queen physiology. PLoS One. 2007;2: e980. doi: 10.1371/journal.pone.0000980 17912357

51. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002;30: 207–10. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=99122&tool=pmcentrez&rendertype=abstract 11752295

52. Elsik CG, Worley KC, Bennett AK, Beye M, Camara F, Childers CP, et al. Finding the missing honey bee genes: lessons learned from a genome upgrade. BMC Genomics. BMC Genomics; 2014;15: 86. doi: 10.1186/1471-2164-15-86 24479613

53. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25: 1105–11. doi: 10.1093/bioinformatics/btp120 19289445

54. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11: R25. doi: 10.1186/gb-2010-11-3-r25 20196867

55. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. BioMed Central Ltd; 2010;11: R106. doi: 10.1186/gb-2010-11-10-r106 20979621

56. Huang DW, Sherman BT, Lempicki R a. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4: 44–57. doi: 10.1038/nprot.2008.211 19131956

57. Supek F, Bošnjak M, Škunca N, Šmuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One. 2011;6: e21800. doi: 10.1371/journal.pone.0021800 21789182

58. Xi Y, Li W. BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics. 2009;10: 232. doi: 10.1186/1471-2105-10-232 19635165

59. Lyko F, Foret S, Kucharski R, Wolf S, Falckenhayn C, Maleszka R. The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol. 2010;8: e1000506. doi: 10.1371/journal.pbio.1000506 21072239

60. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. Nature Publishing Group; 2009;462: 315–22. doi: 10.1038/nature08514 19829295

61. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B …. 1995;

62. Elango N, Hunt BG, Goodisman M a D, Yi S V. DNA methylation is widespread and associated with differential gene expression in castes of the honeybee, Apis mellifera. Proc Natl Acad Sci U S A. 2009;106: 11206–11. doi: 10.1073/pnas.0900301106 19556545

63. Lockett G a, Kucharski R, Maleszka R. DNA methylation changes elicited by social stimuli in the brains of worker honey bees. Genes Brain Behav. 2012;11: 235–42. doi: 10.1111/j.1601-183X.2011.00751.x 22098706

64. Xu J, Grant G, Sabin LR, Gordesky-Gold B, Yasunaga A, Tudor M, et al. Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila. Cell Host Microbe. Elsevier Inc.; 2012;12: 531–43. doi: 10.1016/j.chom.2012.08.011 23084920

65. Kingsolver MB, Huang Z, Hardy RW. Insect antiviral innate immunity: pathways, effectors, and connections. J Mol Biol. Elsevier Ltd; 2013;425: 4921–36. doi: 10.1016/j.jmb.2013.10.006 24120681

66. Mukherjee S, Hanley K a. RNA interference modulates replication of dengue virus in Drosophila melanogaster cells. BMC Microbiol. 2010;10: 127. doi: 10.1186/1471-2180-10-127 20420715

67. Niu J, Meeus I, Cappelle K, Piot N, Smagghe G. The immune response of the small interfering RNA pathway in the defense against bee viruses. Curr Opin Insect Sci. Elsevier Inc; 2014; 22–27. doi: 10.1016/j.cois.2014.09.014 25401084

68. Evans JD, Aronstein K, Chen YP, Hetru C, Imler J-L, Jiang H, et al. Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol. 2006;15: 645–56. doi: 10.1111/j.1365-2583.2006.00682.x 17069638

69. Richard F-J, Holt HL, Grozinger CM. Effects of immunostimulation on social behavior, chemical communication and genome-wide gene expression in honey bee workers (Apis mellifera). BMC Genomics. BMC Genomics; 2012;13: 558. doi: 10.1186/1471-2164-13-558 23072398

70. Holt HL, Aronstein K a, Grozinger CM. Chronic parasitization by Nosema microsporidia causes global expression changes in core nutritional, metabolic and behavioral pathways in honey bee workers (Apis mellifera). BMC Genomics. 2013;14: 799. doi: 10.1186/1471-2164-14-799 24245482

71. Sarda S, Zeng J, Hunt BG, Yi S V. The evolution of invertebrate gene body methylation. Mol Biol Evol. 2012;29: 1907–16. doi: 10.1093/molbev/mss062 22328716

72. Wang Y, Leung FCC. In silico prediction of two classes of honeybee genes with CpG deficiency or CpG enrichment and sorting according to gene ontology classes. J Mol Evol. 2009;68: 700–5. doi: 10.1007/s00239-009-9244-3 19466376

73. Foret S, Kucharski R, Pittelkow Y, Lockett G a, Maleszka R. Epigenetic regulation of the honey bee transcriptome: unravelling the nature of methylated genes. BMC Genomics. 2009;10: 472. doi: 10.1186/1471-2164-10-472 19828049

74. Wang X, Wheeler D, Avery A, Rago A, Choi J-H, Colbourne JK, et al. Function and evolution of DNA methylation in Nasonia vitripennis. PLoS Genet. 2013;9: e1003872. doi: 10.1371/journal.pgen.1003872 24130511

75. Zeng J, Yi S V. DNA methylation and genome evolution in honeybee: gene length, expression, functional enrichment covary with the evolutionary signature of DNA methylation. Genome Biol Evol. 2010;2: 770–80. doi: 10.1093/gbe/evq060 20924039

76. Hellman A, Chess A. Gene body-specific methylation on the active X chromosome. Science. 2007;315: 1141–3. doi: 10.1126/science.1136352 17322062

77. Jones P a. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. Nature Publishing Group; 2012;13: 484–92. doi: 10.1038/nrg3230 22641018

78. Duret L, Mouchiroud D. Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis. Proc Natl Acad Sci U S A. 1999;96: 4482–7. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=16358&tool=pmcentrez&rendertype=abstract 10200288

79. Park J, Xu K, Park T, Yi S V. What are the determinants of gene expression levels and breadths in the human genome? Hum Mol Genet. 2012;21: 46–56. doi: 10.1093/hmg/ddr436 21945885

80. Ehrlich LS, Medina GN, Khan MB, Powell MD, Mikoshiba K, Carter C a. Activation of the inositol (1,4,5)-triphosphate calcium gate receptor is required for HIV-1 Gag release. J Virol. 2010;84: 6438–51. doi: 10.1128/JVI.01588-09 20427533

81. Hrincius ER, Dierkes R, Anhlan D, Wixler V, Ludwig S, Ehrhardt C. Phosphatidylinositol-3-kinase (PI3K) is activated by influenza virus vRNA via the pathogen pattern receptor Rig-I to promote efficient type I interferon production. Cell Microbiol. 2011;13: 1907–19. doi: 10.1111/j.1462-5822.2011.01680.x 21899695

82. Liu Z, Tian Y, Machida K, Lai MMC, Luo G, Foung SKH, et al. Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry. J Biol Chem. 2012;287: 41922–30. doi: 10.1074/jbc.M112.414789 23095753

83. Contreras X, Mzoughi O, Gaston F, Peterlin MB, Bahraoui E. Protein kinase C-delta regulates HIV-1 replication at an early post-entry step in macrophages. Retrovirology. BioMed Central Ltd; 2012;9: 37. doi: 10.1186/1742-4690-9-37 22554282

84. Xu W, Santini P a, Sullivan JS, He B, Shan M, Ball SC, et al. HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits. Nat Immunol. Nature Publishing Group; 2009;10: 1008–17. doi: 10.1038/ni.1753 19648924

85. Strong JE, Coffey MC, Tang D, Sabinin P, Lee PW. The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus. EMBO J. 1998;17: 3351–62. doi: 10.1093/emboj/17.12.3351 9628872

86. Lockett G a, Helliwell P, Maleszka R. Involvement of DNA methylation in memory processing in the honey bee. Neuroreport. 2010;21: 812–6. doi: 10.1097/WNR.0b013e32833ce5be 20571459

87. Park J, Peng Z, Zeng J, Elango N, Park T, Wheeler D, et al. Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes. Mol Biol Evol. 2011;28: 3345–54. doi: 10.1093/molbev/msr168 21693438

88. Shukla S, Kavak E, Gregory M, Imashimizu M, Shutinoski B, Kashlev M, et al. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature. Nature Publishing Group; 2011;479: 74–9. doi: 10.1038/nature10442 21964334

89. Herb BR, Wolschin F, Hansen KD, Aryee MJ, Langmead B, Irizarry R, et al. Reversible switching between epigenetic states in honeybee behavioral subcastes. Nat Neurosci. Nature Publishing Group; 2012;15: 1371–3. doi: 10.1038/nn.3218 22983211

90. Castorena KM, Stapleford KA, Miller DJ. targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication. 2010;